Diabetes is one of the major health challenges in developing countries, which has significant effects on different organs including eye. Although retina is the main ocular target of diabetes, 50%-70% of diabetic patients have corneal abnormalities, which can often be sight threatening. Corneal abnormalities include epithelial defects and fragility, recurrent epithelial erosions, decreased sensitivity, abnormal wound repair, increased susceptibility to injury, ulcers, and edema. We have previously found that beside central cornea, diabetes affects limbus, the junctional zone between cornea and conjunctiva housing corneal epithelial stem cells. Currently, there is a need to better understand the mechanisms responsible for these abnormalities and develop a specific and efficacious treatment. Recently discovered regulatory genetic elements called microRNAs are thought to have broad influences on cell regulation including corneal epithelial cells. We asked whether these microRNAs are engaged in the diabetic state of the cornea and what are the mechanisms of this engagement. Our project seeks to establish if the levels of these microRNAs are altered in the epithelial cells of diabetic corneas by measuring them using novel state-of-the-art deep sequencing methods. Further, to examine their roles and mechanisms of actions, we will utilize adenovirus-driven microRNA-based gene therapy to treat human organ-cultured human autopsy diabetic corneas using functionally validated miRNAs. For the treatment, we will focus on microRNAs that regulate wound healing and can influence the expression of stem cell markers, altered in diabetes. The significance of our study is as follows: (1) The use of deep sequencing method for the first time in the eye, which allows for a discovery of novel microRNAs and to create a comprehensive microRNA profiles in corneal epithelial cells; (2) Precise measurement of differentially produced microRNAs in diabetic cornea, which has not been reported; (3) Translational development of microRNA-based gene therapy for diabetic cornea, which may apply to various corneal disorders; (4) Novel way of normalizing dysfunctional epithelial stem cell in diabetic corneas using microRNA manipulation. We will target diabetic corneal epithelial cells by single microRNAs or their combinations to improve function of corneal epithelium. As a result, this strategy would increase our understanding of microRNA roles in corneal epithelial function as well as in diabetic disease state, which may lead to novel therapy. We will use human corneal organ culture in our studies. Normal and diabetic human autopsy eyes will be purchased from the National Disease Research Interchange (NDRI), a national supplier of donor human research tissue. All tissues are obtained in our lab without donor identity. PUBLIC HEALTH RELEVANCE: This proposal seeks to identify microRNAs differentially expressed in human diabetic corneas and directionally change their levels by gene therapy to normalize diabetes- compromised corneal epithelial wound healing and stem cell marker expression. This would help us to understand the role of microRNAs in regulating epithelial cell functions in normal corneas and diabetic corneal disease. Manipulating the expression of these microRNAs may allow us to ameliorate the diabetic corneal abnormalities, which could contribute to preventing visual impairment associated with diabetes.